JP4637838B2 - Ligation method - Google Patents

Abstract

A method of ligating two or more molecules, for example, peptides to peptides or peptides to labels is provided. The method may comprise the steps: a) providing a first oligopeptide having a reactive moiety, which is a hydrazine moiety, a hydrazide moiety or an amino-óxy moiety b) providing a second oligopeptide having an activated ester moiety, c)allowing the reactive moiety of the first oligopeptide to react with the activated ester moiety of the second oligopeptide to form an oligopeptide product, in which the first and second oligopeptides are linked via a linking moiety having Formula I, II or III. The second oligopeptide may preferably be generated by thiol reagent induced cleavage of an intein fusion protein. The invention further provides labelling and ligation methods in which protein hydrazides are ligated by reaction of the hydrazide moiety with an aldehyde functionality or a ketone functionality.

Description

Field of Invention

  The present application relates to a method of linking two or more molecules, such as small organic molecules, labels, peptides, and the like. In particular, this application relates to methods for linking peptides, such as ligation of synthetic peptides to recombinant peptides.

Background of the Invention

  Protein engineering methods have proven to be very valuable for basic research applications, diagnostics, drug discovery, and for creating protein-based tools as protein therapeutics. The ability to manipulate the primary structure of proteins in a controlled manner opens many new possibilities in biological science and medicine. Therefore, there is a concerted effort to develop methods for site-specific modification of proteins and then application of them.

  The two main methods of protein production are by recombinant methods or chemical synthesis. So far, these two methods have been found to be complementary. That is, recombination methods can produce proteins of any size, but they are usually limited to the assembly of amino acids derived from proteins. Therefore, in general, labeling and probe introduction into a recombinant protein must be performed after translation and cannot be modified into the protein backbone.

The most common method for labeling recombinant proteins uses an amino or thiol reactive type of label that reacts covalently with the lysine side chain / N ^alpha amino group or cysteine side chain inside the protein, respectively. To do. In order for such a labeling method to be site specific, an appropriate protein derivative containing a reactive functional group specific to the position to be modified must be designed. This requires that all other naturally reactive functional groups in the primary sequence are removed by amino acid mutagenesis. In the case of protein amino functional groups, this is almost impossible due to the large amount of lysine residues inside the protein and the amino functional group present at the N-terminus of the sequence. Similarly, in the case of cysteine, this process is labor intensive and often detrimental to protein function.

  Production of proteins with site-specific modifications and / or labels can be more easily achieved using chemical synthesis methods. Chemical synthesis of proteins allows multiple modifications to be incorporated site-specifically in both the side-chain and main-chain portions of the protein, but usually the largest of the sequences that can be synthesized and isolated The size is about 50-100 amino acids.

Protein ligation Another method of protein production is protein / peptide ligation. In this method, mutually reactive chemical functional groups (orthogonals that do not interact with the chemical properties of the natural amino acid, ie, due to the mutually exclusive chemical properties compared to the reaction of the reactive portion of the natural amino acid). Reacts) at the N- and C-termini of unprotected polypeptide fragments so that when they are mixed, they react chemoselectively to join the two sequences together ( Cotton GJ and Muir TW. Chem. Biol., 1999, 6, R247-R254). The principle of chemical ligation is shown schematically in FIG.

  Several chemistries have been utilized to perform ligation of two synthetic peptides, and a diverse range of different chemical functional groups can be incorporated at the end of a polypeptide by solid phase peptide synthesis. These include reactions of thioacids with bromoalkyl to form thioesters (Schnolzer M and Kent SBH, Science, 1992, 256, 221-225), aldehydes to form thiazolidine or oxazolidine, respectively. And N-terminal cysteine or threonine (Liu C-F and Tam JP Proc. Natl. Acad. Sci. USA, 1994, 91, 6584-6588), hydrazides and aldehydes to form hydrazones (Gaertner HF et al., Bioconj. Chem., 1992, 3, 262-268), reaction of an aminoxy group with an aldehyde to form an oxime (Rose K. J. Am. Chem. So. 1994, 116, 30-33), reaction of azide with aryl phosphine to form an amide bond (Staudinger ligation) (Nilsson BL, Kiessling LL, and Raines RT. Org.). Lett., 2001, 3, 9-12, Kiick et al. Proc. Natl. Acad. Sci. USA, 2002, 99, 19-24), and the C-terminus to form a natural amide bond. Reaction of a peptide having a thioester with a peptide having a cysteine at the N-terminus (Dawson et al. Science, 1994, 266, 776) (Native Chemical Ligation Method, US Pat. No. 6,184,344, EP0832096B1)This native chemical ligation method is based on studies by Wieland and co-workers who have shown that reaction of ValSPh and CysOH in aqueous buffer yields the dipeptide ValCysOH (Wieland T et al., Liebigs Ann. Chem., 1953, 583 129-149).

  Native chemical ligation methods have become popular, but this requires a peptide containing a cysteine at the N-terminus for the reaction, so if the cysteine is not present at the proper position in the protein, Cysteine must be introduced into the ligation site. However, introducing extra thiol groups into the protein sequence, in particular cysteine, has a tendency to form disulfide bonds that can inhibit the folding pathway or impair the function of the folded protein. May be harmful to function.

  Because of the difficulties and problems associated with known ligation techniques, ligation of two synthetic fragments usually only allows chemical synthesis of proteins consisting of about 100-150 amino acids. Larger proteins have been synthesized by linking two or more fragments together, but this has proven to be technically difficult (Camarero et al. J. Pept. Res., 1998, 54, 303-316, Canne LE et al., J. Am. Chem. Soc., 1999, 121, 8720-8727).

Protein Semi-Synthesis Protein ligation techniques are described that can bind synthetic protein fragments or recombinantly derived protein fragments to each other. This allows large proteins to be constructed from a combination of synthetic and recombinant fragments, and allows the protein to be modified site-specifically, both natural and non-natural. By utilizing such so-called protein semisynthesis, many different synthetic moieties can be incorporated site-specifically at a plurality of different sites within the target protein.

  In order to utilize a recombinant protein in a ligation strategy, the recombinant fragment must contain an appropriate reactive functional group to facilitate ligation. One method for introducing a unique reactive functional group into a recombinant protein is by periodate oxidation of an N-terminal serine-containing sequence. Such treatment converts the N-terminal serine to a glyoxyl moiety containing an N-terminal aldehyde. Next, synthetic peptides containing hydrazides were ligated to the N-terminus of these proteins, chemoselectively through formation of hydrazone bonds with proteins having a glyoxyl group at the N-terminus (Gaertner HF et al., Bioconj. Chem., 1992, 3, 262-268, Gaertner HF et al., J. Biol. Chem., 1994, 269, 7224-7230). Another effort has been made to generate recombinant proteins with an N-terminal cysteine residue. Subsequently, synthetic peptides containing thioesters at the C-terminus were site-specifically linked to the N-terminus of these proteins through amide bond formation in a manner similar to the “native chemical ligation method” (Cotton GJ and Muir TW. Chem. Biol., 2000, 7, 253-261). However, similar to ligation of synthetic peptides by the native chemical ligation method, this technique requires the introduction of a cysteine at the ligation site if the primary sequence does not contain a cysteine at the appropriate position.

Protein Splicing Technology Recently, a technology has been developed that enables the production of recombinant proteins containing a C-terminal thioester group. The C-terminal thioester functionality provides a unique reactive chemical group within the protein that can be utilized for protein ligation. Recombinant C-terminal thioester proteins are made by manipulating a natural biological phenomenon known as protein splicing (Pauls H. Annu Rev Biochem 2000, 69, pp 447-496). Protein splicing is a post-translational process in which protein precursors undergo a series of intramolecular rearrangements that result in the precise removal of an internal region called intein and the ligation of two flanking sequences called exteins (FIG. 2). In general, neither extein has sequence requirements, but inteins are characterized by several conserved sequence motifs, and well over 100 members of this protein domain family have now been identified.

  The first step of protein splicing involves transferring the N-terminal extein unit to the SH or OH group of the side chain of the conserved residue Cys / Ser / Thr that is always located at the adjacent N-terminus of the intein. With N → S (or N → O) acyl rearrangement. From this mechanistic insight, several mutant inteins have been designed that can only promote the first step of protein splicing (Chong et al. Gene. 1997, 192, 271-281, (Noren et al., Angew. Chem. Int. Ed. Engl., 2000, 39, 450-466) proteins expressed as in-frame N-terminal fusions to one of these artificially designed inteins Cleavage by a thiol via a transthioesterification reaction can generate a C-terminal thioester recombinant protein derivative (FIG. 3) (Chong et al. Gene. 1997, 192, 271-281, (Noren et al., Angew). Chem.Int.Ed.Engl., 2000, 39, pages 450-466) (New England Biolabs Impact System International Publication No. 00/18881, International Publication No. 0047751) Next, expressed protein ligation (expressed protein ligation, EPL) or intein-mediated protein ligation (EPL) The peptide sequence containing the N-terminal cysteine residue can be specifically ligated to the C-terminus of such a recombinant C-terminal thioester protein by a procedure called intein-mediated protein ligation (IPL) (Mir et al. Proc. Natl.Acad.Sci.USA, 1998, 95, 6705-6710, Evans Jr et al. Prot.Sci., 1998, , Pp. 2256 to 2264).

  Chemoselective ligation of N-terminal cysteine-containing peptides to peptides containing C-terminal thioesters, whether synthetic or recombinant, is generally performed in the presence of a thiol cofactor at slightly basic pH. . This strategy also requires the introduction of a cysteine at the ligation site if the cysteine is not properly located in the primary sequence. These conditions of this ligation method have the potential to alter the structure and / or function of both the protein ligation product and the initial reactant.

  For example, the chemokine RANTES is 100 mM 2-mercaptoethanesulfonic acid, a buffer commonly used to perform ligation of C-terminal thioester molecules (expressed protein ligation and native chemical ligation) to molecules containing an N-terminal cysteine. It is unstable in a buffer solution of pH 7.4 consisting of 100 mM NaCl and 100 mM sodium phosphate containing (MESNA). RANTES has two disulfide bonds that are important for maintaining protein structure and function. In the standard ligation buffer described above, it was found that the folded protein was converted to a mixture of reduced protein and MESNA protein adduct within 48 hours. The majority of the protein mixture probably formed a precipitate afterwards, probably reflecting the unfolded nature of these species (Cotton, unpublished).

  Thus, we have ligated reactions that require thiol-containing buffers generally to maintain the integrity of antibodies, antibody fragments, and proteins with disulfide bonds, such as antibody domains, cytokines, growth factors, etc. I think that it is not suitable. Therefore, there is a need for a ligation method that is usually performed in the absence of thiols. For example, when observed over several days, RANTES was found to be stable in a pH 7.4 buffer consisting of 100 mM NaCl and 100 mM sodium phosphate, and a 100 mM sodium acetate buffer at pH 4.5. Unpublished results). Thus, ligation reactions that can be performed under such conditions should be applicable to proteins with and without disulfides.

Protein labeling Historically, protein ligation means that two peptides / protein fragments are linked together, which is synonymous with protein labeling, where the label is a peptide or derivatized peptide. Similarly, if the non-peptidic synthetic small molecule contains the reactive chemical functional group necessary for protein ligation, ligating the synthetic molecule directly to either the N-terminus or C-terminus of the protein, The protein is labeled site-specifically. Therefore, the technology developed for ligation of protein fragments should be used to directly label either its N-terminus or C-terminus in a site-specific manner independent of the peptide or protein sequence. You can also.

  A recombinant protein containing an N-terminal glyoxyl functional group (generated by periodate oxidation of the corresponding N-terminal serine protein) labels the N-terminus in a site-specific manner by reaction with a labeled hydrazide or aminoxy derivative. (Geoghegan KF and Stroh JG. Bioconj Chem., 1992, 3, 138-146, Alouni S et al. Eur. J. Biochem., 1995, 227, 328-334). Recombinant proteins containing an N-terminal cysteine residue are also labeled with thioester functional groups and thioester acyl substituents to form amides and thiazolidines, respectively (Schuler B and Pannel LK. Bioconjug. Chem. , 2002, 13, 1039-43), and through reaction with aldehyde functionality (Zhao et al. Bioconj. Chem., 1999, 10, 424-430).

  There are several methods for ligating proteins, but each method has potential drawbacks. Therefore, new ligation methods are needed, especially for ligation of proteins that are compatible with synthetic and recombinant fragments and that have disulfide bonds as well as proteins that do not have disulfide bonds. There is a need for a ligation method that complements existing technology and provides another means for protein engineers.

Summary of the Invention

  The inventors have developed a new method for linking peptide molecules that overcomes some of the problems associated with the prior art and overcomes some of the problems of the prior art.

Therefore, in the first aspect of the present invention,
a) providing a first oligopeptide having a reactive moiety;
b) providing a second oligopeptide having an activated ester moiety;
c) reacting the reactive moiety of the first oligopeptide with the activated ester moiety of the second oligopeptide, via the linking moiety of formula I, formula II, or formula III Forming an oligopeptide product in which a peptide and a second oligopeptide are linked, a method of making an oligopeptide product is provided.

  In a preferred embodiment, the oligopeptide is linked in step (c) via a linking moiety represented by formula II, and the activated ester moiety in step (b) is not a thioester, and the activated ester is , The terminal activated ester moiety.

  In a further preferred embodiment of the present invention, the connecting portion is connected via a connecting portion represented by Formula I or Formula III.

  Unless the context requires otherwise, the terms peptide, oligopeptide, polypeptide, and protein are used interchangeably.

  The activated ester moiety of the first oligopeptide may be any suitable activated ester moiety such as a thioester moiety, a phenol ester moiety, a hydroxysuccinimide moiety, or an O-acylisourea moiety.

  In a preferred embodiment of the invention, the activated ester moiety is a thioester moiety. Any suitable thioester peptide where the peptide is an acyl substituent of a thioester may be used in the present invention (FIG. 4).

  Such thioester peptides may be produced synthetically or recombinantly. Those skilled in the art are familiar with methods known in the art for producing synthetic peptide thioesters. For example, synthetic peptide thioesters can be made by synthesis on a resin that cleaves HF and at the same time yields a C-terminal thioester (Hojo et al., Bull. Chem. Soc. Jpn., 1993, 66, 2700-2706. page). In addition, the use of “safety catch” linkers is widely used to generate C-terminal thioesters by cleaving assembled peptides from thiol-introducing resins (Shin Y et al., J. Am. Chem. Soc. 1999, 121, 11684-11689).

  In addition, recently, techniques have been developed that allow the production of recombinant C-terminal thioester proteins. Recombinant C-terminal thioester proteins can be made by manipulating a natural biological phenomenon known as protein splicing. As mentioned above, protein splicing is a post-translational process in which protein precursors undergo a series of intramolecular rearrangements to accurately remove an internal region called intein and two flanking sequences called exteins. It is a process that brings about ligation.

  As mentioned above, several mutant inteins have been designed that can only promote the first step of protein splicing (Chong et al. Gene. 1997, 192, 271-281, Noren et al., Angew. Chem. Int. Ed. Engl., 2000, 39, 450-466). A protein expressed as an in-frame N-terminal fusion to one of these artificially designed inteins is cleaved by a thiol via an intermolecular transthioesterification reaction to produce a C-terminal thioester recombinant protein derivative (Chong et al. Gene. 1997, 192, 271-281, Noren et al., Angew. Chem. Int. Ed. Engl., 2000, 39, 450-466) (New England Biolabs Impact) System International Publication No. 00/18881, International Publication No. 0047751). Such protein thioesters may be used in the methods of the invention (see FIG. 3).

  Accordingly, in a preferred embodiment of the present invention, in step (b), a second oligopeptide is generated by intein fusion protein cleavage induced by a thiol reagent.

Therefore, in the second aspect of the present invention,
a) providing a first oligopeptide having a reactive moiety;
b) (i) providing an oligopeptide precursor molecule comprising a second oligopeptide precursor fused to the intein domain at the N-terminus;
(Ii) causing the precursor molecule to undergo thiol reagent-dependent cleavage to produce a second oligopeptide molecule having a thioester moiety at the C-terminus;
c) reacting the reactive part of the first oligopeptide with the second oligopeptide molecule, via the linking moiety of formula I, II, or III, the first oligopeptide and the second oligo Forming an oligopeptide product with which the peptide is linked is provided.

The reactive moiety of the first oligopeptide may be any suitable reactive moiety. In a preferred embodiment of the invention, the reactive moiety is a hydrazine moiety, an aminooxy moiety, or a hydrazide moiety represented by the general formula IV, V, or VI, respectively.

  For example, in certain preferred embodiments, the reactive moiety is represented by Formula IV, and in the oligopeptide product made by the method of the present invention, the first oligopeptide and the second oligopeptide are represented by Formula I: Are connected via a connecting portion.

  In a further preferred embodiment, the reactive moiety is represented by formula V, and in the oligopeptide product made by the method of the invention, the first oligopeptide and the second oligopeptide are linked by the formula II It is connected through parts.

  In another preferred embodiment, the reactive moiety is represented by Formula VI, and in the oligopeptide product made by the method of the invention, the first oligopeptide and the second oligopeptide are represented by Formula III It is connected via a connecting part.

  As described above, the first oligopeptide includes a reactive moiety, which in a preferred embodiment is a hydrazine moiety (eg, Formula IV), an aminooxy moiety (eg, Formula V), or a hydrazide moiety (eg, Formula VI). It's okay.

  A particular advantage of the ligation method of the present invention is that it can be performed in the absence of thiols. This allows efficient ligation of proteins / peptides containing disulfide bonds as well as proteins without such bonds.

  Accordingly, in embodiments of the first and second aspects of the invention, at least one of the first oligopeptide and the second oligopeptide has one or more disulfide bonds.

  Derivatives of synthetic oligopeptides containing hydrazine, hydrazide, or aminooxy can be readily prepared using known methods such as solid phase synthesis.

  Furthermore, the present inventors also discovered that a protein fused to the intein domain at the N-terminus can be selectively cleaved from the intein by hydrazine treatment to release the desired protein as the corresponding hydrazide derivative. (See, eg, FIG. 5).

  Thus, in a further preferred embodiment of the invention, the first oligopeptide is produced by reaction of hydrazine with an oligopeptide molecule comprising a first oligopeptide fused to the intein domain at the N-terminus.

  Indeed, the discovery that such a reaction form can produce such a protein hydrazide has become an independent aspect of the present invention.

Therefore, the third aspect of the present invention
a) providing a protein molecule comprising an oligopeptide fused to the intein domain at the N-terminus;
b) reacting the protein molecule with hydrazine such that the intein domain is cleaved from the oligopeptide to produce a protein hydrazide.
A method for producing a protein hydrazide is provided.

  Further, in addition to using such a reaction to generate a first oligopeptide having a hydrazide moiety at the C-terminus, as described above, the first oligopeptide has a second oligopeptide having an activated ester moiety. Because it can be utilized for reaction with peptides, the present invention further extends to a “one-step” method for linking two peptides to produce an oligopeptide product.

  This can be achieved by reacting a suitable protein with the N-terminus directly attached to the intein with a polypeptide having a hydrazine, hydrazide, or aminooxy moiety.

Thus, in a fourth aspect, the present invention provides
a) providing a first oligopeptide having a reactive moiety that is a hydrazine moiety, a hydrazide moiety, or an aminooxy moiety;
(I) providing an oligopeptide precursor molecule comprising a second oligopeptide fused to the intein domain at the N-terminus;
c) reacting the reactive portion of the first oligopeptide with the oligopeptide precursor molecule to form the first oligopeptide and the second via a linking moiety of formula I, formula II, or formula III; Forming an oligopeptide product linked to the oligopeptide,
Methods for making oligopeptide products are provided.

  Thus, the ligation technique of the present invention can utilize both synthetic proteins and peptides as well as recombinant proteins and peptides. Accordingly, this allows ligation of two or more synthetic peptides, ligation of two or more recombinant peptides, or ligation of at least one synthetic peptide and at least one recombinant peptide.

  Furthermore, in addition to providing a novel method for linking peptides, the present invention can also be used to label synthetic or recombinant peptides.

Therefore, in the fifth aspect of the present invention,
a) providing a labeled molecule having a reactive moiety;
b) providing an oligopeptide having an activated ester moiety;
c) reacting the reactive part of the label molecule with the activated ester part of the oligopeptide and via the linking moiety of formula I, formula II or formula III as defined above, Forming a labeled oligopeptide linked to
Methods are provided for labeling oligopeptides.

  In a preferred embodiment, in step (c), the labeled molecule and the oligopeptide are linked via a linking moiety represented by formula II, wherein the activated ester moiety in step (b) is not a thioester and the active The activated ester is the terminal activated ester moiety.

  In a preferred embodiment of the invention, in step (b), an oligopeptide is generated by thiol-induced intein fusion protein cleavage.

Therefore, in the sixth aspect of the present invention,
a) providing a labeled molecule having a reactive moiety;
c) (i) providing an oligopeptide precursor molecule comprising an oligopeptide precursor fused to the intein domain at the N-terminus;
(Ii) causing thiol reagent-dependent cleavage of the precursor molecule to produce an oligopeptide molecule having a thioester moiety at the C-terminus;
c) reacting the reactive part of the labeled molecule with the oligopeptide to produce a labeled oligopeptide in which the labeled molecule and the oligopeptide are linked via a linking moiety of formula I, II or III Forming a step,
Methods are provided for labeling oligopeptides.

Alternatively, an oligopeptide having a reactive moiety can be labeled with a label molecule having an activated ester moiety at the end. Therefore, in the seventh aspect of the present invention,
a) providing a labeled molecule having an activated ester moiety that is an acyl substituent;
b) providing an oligopeptide having a reactive moiety;
c) reacting the activated ester moiety of the labeled molecule with the reactive moiety of the oligopeptide to link the labeled molecule to the oligopeptide via a linking moiety represented by Formula I, Formula II, or Formula III. Forming a labeled oligopeptide comprising:
A method for labeling oligopeptides, comprising:
In step (c), the labeled molecule and oligopeptide are linked via a linking moiety represented by formula II, wherein the activated ester moiety in step (b) is not a thioester and the activated ester is terminal activated. Methods are provided that are ester moieties.

Similar to the ligation technique, oligopeptides present as precursor molecules linked to intein molecules can also be directly labeled. Accordingly, the eighth aspect of the present invention provides
a) providing a labeled molecule having a reactive moiety;
b) providing an oligopeptide precursor molecule comprising an oligopeptide fused to the intein domain at the N-terminus;
c) The reactive part of the label molecule is reacted with the oligopeptide precursor molecule, and the label molecule and the oligopeptide are linked via the linking moiety represented by formula I, formula II, or formula III as defined above. Forming a labeled oligopeptide product, wherein
Methods for labeling oligopeptides are provided.

  Any suitable label molecule known to those skilled in the art may be used in the methods of the invention. The choice of label will depend on the application for which the label peptide is to be used. For example, labels that can be used in the method of the present invention include fluorophores, cross-linking reagents, spin labels, affinity probes, imaging reagents such as radioisotopes, chelating agents such as DOTA, polymers such as PEG, lipids, sugars, cytotoxicity Mention may be made of drugs and solid surfaces and beads.

  In certain embodiments of the fifth, sixth, and seventh aspects of the invention, at least one of the label and the oligopeptide has one or more disulfide bonds.

  The methods of the invention are particularly useful in peptide ligation, particularly peptide ligation that would require various protecting groups using conventional ligation techniques. The inventors have shown that the method of the invention can be carried out under pH conditions where only the reactive moiety reacts.

  In preferred embodiments of the first and second aspects of the invention, and in preferred embodiments of the fourth to eighth aspects of the invention, a range of pH 4.0 to pH 8.5, preferably pH 4.0 to pH 8. 0, for example, pH 4.0 to 7.5, more preferably pH 5.0 to pH 8.0, more preferably pH 6.0 to pH 7.5, most preferably pH 6.5 to pH 7.5. Perform step (c) of the process at a pH in the range.

  For example, the inventors have demonstrated that the C-terminal thioester of a synthetic peptide reacts specifically with hydrazine under aqueous conditions at pH 6.0 to form the corresponding peptide hydrazide. This allows the ligation method described herein to be performed at pH 6.0 without the need for potentially harmful thiol cofactors (useful if the fragment or final construct is sensitive to thiols). And the introduction of potentially reactive side chain groups (such as thiols) into the protein does not occur. Similarly, we may also note that the C-terminal thioester of a synthetic peptide reacts specifically with hydroxylamine under aqueous conditions at pH 6.0 and pH 6.8 to form the corresponding peptide hydroxamic acid. Demonstrated. Furthermore, as described below, the present inventors specifically reacted with O-methylhydroxylamine at pH 7.5, aqueous conditions, both the synthetic peptide C-terminal thioester and the recombinant protein C-terminal thioester, It has also been demonstrated to form the corresponding C-terminal N-methoxyamide derivative. This allows the ligation method described herein to be performed at pH 7.5 without the need for potentially harmful thiol cofactors.

  Peptides and proteins containing a thioester group (where the peptide is an acyl substituent of the thioester) are reacted with a hydrazine, hydrazide, or aminooxy derivative of the label or peptide to provide site-specific labeling and chemoselective ligation, respectively. Can be performed (see, eg, FIGS. 4 and 5).

  In a similar manner, peptides containing hydrazine, hydrazide, or aminooxy groups can be reacted with a label or a thioester derivative of the peptide for site-specific labeling and chemoselective ligation, respectively (eg, FIG. 4 and See FIG.

  In addition, the inventors have demonstrated that the protein-intein fusion can be cleaved with hydrazine to produce a recombinant protein hydrazide, and the hydrazide moiety can be a reactive group other than an activated ester moiety, such as It has also been shown that such protein hydrazides can be linked by reacting with aldehyde functional groups or ketone functional groups. For example, as described below, the inventors have shown that the described method can be used to chemoselectively link a pyruvyl derivative of a synthetic peptide to the C-terminus of a recombinant protein hydrazide, and in a similar manner, By the method described, the C-terminus of the recombinant protein hydrazide was site-specifically labeled with a pyruboyl derivative of fluorescein.

  This aspect of the invention provides a further novel method of linking a recombinant peptide to a second peptide or indeed to a label.

Accordingly, the ninth aspect of the present invention provides
a) providing a first oligopeptide having an aldehyde or ketone moiety;
b) providing an oligopeptide precursor molecule comprising a second oligopeptide fused to the intein domain at the N-terminus;
c) reacting the oligopeptide precursor molecule with hydrazine to produce an oligopeptide molecule comprising an oligopeptide intermediate having a hydrazide moiety at the C-terminus;
d) The first oligopeptide and the second oligopeptide are linked via a hydrazone linking moiety by reacting the aldehyde or ketone moiety of the first oligopeptide with the hydrazide moiety of the oligopeptide intermediate molecule. Forming an oligopeptide product,
Methods for making oligopeptide products are provided.

  An example of this aspect is shown in FIG.

The tenth aspect of the present invention provides
a) providing a labeled molecule having an aldehyde or ketone moiety;
b) providing an oligopeptide precursor molecule comprising a first oligopeptide fused to the intein domain at the N-terminus;
c) reacting said oligopeptide precursor molecule with hydrazine to produce an oligopeptide molecule comprising an oligopeptide intermediate having a hydrazide moiety at the end;
d) reacting the aldehyde or ketone moiety of the labeled molecule with the hydrazide moiety of the oligopeptide intermediate molecule to produce a labeled oligopeptide product in which the labeled molecule and oligopeptide are linked via a hydrazone linking moiety. Forming a step,
Methods for labeling oligopeptides are provided.

（式中、ＲはＨ、または任意の置換もしくは非置換、好ましくは非置換のアルキル基である。） In preferred embodiments of the ninth and tenth aspects of the invention, the hydrazone moiety is represented by Formula VII.

Wherein R is H or any substituted or unsubstituted, preferably unsubstituted alkyl group.

  In preferred embodiments of the ninth and tenth aspects of the present invention, a range of pH 1.0 to pH 7.0, preferably pH 1.0 to pH 6.0, more preferably a range of pH 2.0 to pH 5.5, most The method is preferably carried out at a pH in the range of pH 2.0 to pH 4.5.

  In certain embodiments of the ninth and tenth aspects of the invention, the aldehyde or ketone containing moiety of the oligopeptide or label is an α-diketone group or an α-ketoaldehyde group.

  In an eleventh aspect of the present invention, oligopeptide products made using the methods of the present invention are provided.

  In a twelfth aspect, a labeled oligopeptide comprising an oligopeptide labeled by the method of the present invention is provided.

  Preferred features of each aspect of the invention are modified as necessary for each of the other aspects.

  The invention will now be further described by the following non-limiting examples with reference to the accompanying drawings.

Example 1 Protein Ligation / Site Specific Protein Labeling Using Reactions of Peptide / Protein Thioesters with Hydrazine / Hydrazide or Compounds Containing Aminoxy Functionality A) Peptides with Thioesters at the C-Terminal and 100 mM at pH 6.0 Reaction with Hydrazine 200 mM sodium phosphate buffer at pH 6.0 containing 100 mM hydrazine monohydrate (200 μL) was added to a model synthetic C-terminal thioester peptide (200 μg) termed AS626p1A to give a final peptide concentration of 317 μM. It was. AS626p1A has the sequence ARTKQTARK (Me) ₃ STGGKAPRKQ LATKAARK-COS- (CH ₂ ) ₂ -COOC ₂ H ₅ (SEQ ID NO: 1), in which only one alanine residue (SEQ ID NO: 1) Any one of the alanine residues) is replaced by an arginine residue. The reaction was incubated at room temperature and observed over time using analytical reverse phase HPLC. Vydac C18 column (5 μM, 0.46 × 25 cm). Peptides were eluted at a flow rate of 1 mL / min using a linear gradient of acetonitrile-water / 0.1% TFA. The characteristics of individual peptides eluting from the column were determined by electrospray mass spectrometry.

B) Reaction of a peptide having a thioester at the C-terminus and 100 mM hydroxylamine at pH 6.0 200 mM sodium phosphate buffer at pH 6.0 containing 100 mM hydroxylamine hydrochloride (200 μL) was added to AS626p1A (200 μg), The final peptide concentration was 317 μM. The reaction was incubated at room temperature and observed over time using analytical reverse phase HPLC. Vydac C18 column (5 μM, 0.46 × 25 cm). Peptides were eluted at a flow rate of 1 mL / min using a linear gradient of acetonitrile-water / 0.1% TFA. The characteristics of individual peptides eluting from the column were determined by electrospray mass spectrometry.

C) Reaction of a peptide having a thioester at the C-terminus with 100 mM hydroxylamine at pH 6.8 200 mM sodium phosphate buffer at pH 6.8 containing 100 mM hydroxylamine hydrochloride (200 μL) was added to AS626p1A (200 μg), The final peptide concentration was 317 μM. The reaction was incubated at room temperature and observed over time using analytical reverse phase HPLC. Vydac C18 column (5 μM, 0.46 × 25 cm). Peptides were eluted at a flow rate of 1 mL / min using a linear gradient of acetonitrile-water / 0.1% TFA. The characteristics of individual peptides eluting from the column were determined by electrospray mass spectrometry.

D) Reaction of a peptide having a thioester at the C-terminus with 10 mM hydroxylamine at pH 6.8 The procedure described in C) was repeated, replacing 100 mM hydroxylamine with 10 mM hydroxylamine.

E) Reaction of a peptide having a thioester at the C-terminus with 10 mM hydroxylamine at pH 7.5 The procedure described in D) was repeated at pH 7.5.

F) Reaction of peptide having thioester at C-terminus with 2 mM hydroxylamine at pH 7.5 The procedure described in E) was repeated replacing 10 mM hydroxylamine with 2 mM hydroxylamine.

G) Reaction of a peptide having a thioester at the C-terminus and 100 mM O-methylhydroxylamine (NH ₂ —O—CH ₃ ) at pH 7.5 200 mM at pH 7.5 containing 100 mM O-methylhydroxylamine (200 μL) Sodium phosphate buffer was added to the synthetic peptide AS626p1A (200 μg) whose C-terminus is a thioester to give a final peptide concentration of 317 μM. The reaction was incubated at room temperature and observed over time using analytical reverse phase HPLC. Vydac C18 column (5 μM, 0.46 × 25 cm). Peptides were eluted at a flow rate of 1 mL / min using a linear gradient of acetonitrile-water / 0.1% TFA. The characteristics of individual peptides eluting from the column were determined by electrospray mass spectrometry.

H) Reaction of a peptide having a thioester at the C-terminus with 10 mM O-methylhydroxylamine at pH 7.5. The procedure described in G) was repeated replacing 100 mM O-methylhydroxylamine with 10 mM O-methylhydroxylamine. .

I) Reaction of recombinant protein with thioester at C-terminus and 100 mM O-methylhydroxylamine at pH 7.5 by cleavage of the fusion protein Grb2-SH2-GyrA intein-CBD as described in Example 2 below. The C-terminal mercaptoethanesulfonic acid thioester derivative of recombinant Grb2-SH2 was generated. This recombinant C-terminal thioester protein (100 μg) was reacted with 100 mM O-methylhydroxylamine (200 μL) dissolved in 200 mM sodium phosphate buffer at pH 7.5. The reaction was incubated at room temperature and observed over time using analytical reverse phase HPLC. Vydac C5 column (5 μM, 0.46 × 25 cm). Peptides were eluted at a flow rate of 1 mL / min using a linear gradient of acetonitrile-water / 0.1% TFA. The characteristics of individual peptides eluting from the column were determined by electrospray mass spectrometry.

Results These examples use the reaction of peptide / protein C-terminal thioesters with compounds containing hydrazine / hydrazide or aminoxy functional groups to produce protein ligations / sites of both synthetic and recombinant protein sequences of the invention. It represents a novel strategy for performing specific protein labeling.

  As described above, purified synthetic 27 amino acid C-terminal thioester peptide (ethyl 3-mercaptopropionate thioester derivative) was treated with hydrazine and hydroxylamine under various conditions (Table 1).

  Treatment with pH 6.0, 100 mM hydrazine formed a peptide species that eluted earlier than the starting thioester peptide as analyzed by HPLC. This material was identified by ESMS as the expected peptide hydrazide: observed mass = 3054 Da, expected mass (average of isotope compounds) is 3053 Da. The reaction of a peptide having a C-terminal thioester and hydrazine to form a peptide hydrazide was observed over time using reverse phase HPLC. Even after 3 days, only the desired material was formed without forming by-products. The stability of the peptide hydrazide under these reaction conditions suggests that this reaction occurs at the C-terminal thioester moiety and has chemoselective properties. This also emphasizes that this reaction can be applied for protein ligation and labeling (70% conversion in 2 hours,> 95% conversion in 4 hours).

  To determine if aminooxy-containing compounds react chemoselectively with the peptide / protein C-terminal thioester for protein ligation and site-specific labeling, the synthetic C-terminal thioester peptide is subjected to various conditions under various conditions: Treated with hydroxylamine (Table 1).

Purified synthetic 27 amino acid C-terminal thioester peptide (ethyl 3-mercaptopropionate thioester, observed mass 3155 Da) was incubated at room temperature with various concentrations of hydroxylamine dissolved in aqueous buffer at various pHs. In all cases, as analyzed by reverse phase HPLC, the peptide whose C-terminus was a thioester reacted to form a single product that eluted earlier than the starting thioester peptide. As determined by ESMS, this material corresponds to the expected peptide hydroxamic acid: observed mass = 3052 Da, expected mass (average of isotope compounds) is 3054 Da. Reaction kinetics were observed using reverse phase HPLC. Peptides whose C-terminus is a thioester were converted to the corresponding peptide hydroxamic acid in a clean manner without the formation of by-products. Increasing the pH of the reaction buffer increased the reaction rate. For example, as a result of shifting from pH 6.0 to pH 6.8 using NH ₂ OH at a concentration of 100 mM, the product formation rate after 1 hour increased from 25% to 91%. The reaction rate with 100 mM NH ₂ OH at pH 6.0 was comparable to 10 mM NH ₂ OH at pH 6.8.

The reaction rate of a hydroxylamine with a peptide having a C-terminal thioester to form the corresponding hydroxamic acid increases with increasing pH and decreases with decreasing NH ₂ OH concentration. In order to identify pH and reactant concentration conditions appropriate for peptide / protein labeling and ligation, labeling was performed with increasing pH and decreasing NH ₂ OH concentration.

The reaction with 10 mM NH ₂ OH was 83% complete after 4 hours at pH 6.8, compared to 83% after 2 hours at pH 7.5. Further NH 2 _OH concentrations result was lowered to 2 mM, the reaction rate at pH7.5 is significantly reduced, after 8 hours, 70% of the starting time of the α- thioester peptide was converted to the corresponding hydroxamic acid. It was noted that a small amount of by-product, with a mass corresponding to the peptide acid, was formed during the reaction. This is believed to have been formed by a competitive hydrolysis side reaction at pH 7.5. This side reaction was not observed when using 10 mM NH ₂ OH at pH 7.5, since the reaction occurred faster at this higher reactant concentration.

  To further investigate the chemoselective reaction between aminooxy-containing compounds and peptide / protein C-terminal thioesters for protein ligation and site-specific labeling, the synthetic C-terminal thioester peptide AS626p1 was treated with O-methylhydroxylamine. did.

  Purified synthetic 27 amino acid C-terminal thioester peptide (ethyl 3-mercaptopropionate thioester, observed mass 3155 Da) was incubated at room temperature with 100 mM O-methylhydroxylamine dissolved in 200 mM sodium phosphate buffer at pH 7.5. . As analyzed by reverse phase HPLC, the peptide C-terminal thioester reacted to form a single product that elutes earlier than the starting thioester peptide. As determined by ESMS, this material corresponded to the expected N-methoxy peptide amide: observed mass = 3070 Da, expected mass is 3068 Da. Reaction kinetics were observed using reverse phase HPLC (Table II). The peptide C-terminal thioester was converted to the corresponding N-methoxy peptide amide derivative in a clean manner without by-product formation and 75% of the reaction was complete after 24 hours. Under these conditions, no hydrolysis of the thioester was observed.

  When the reaction was repeated under the same conditions except that 10OmM O-methylhydroxylamine replaced 100mM O-methylhydroxylamine, the reaction rate slowed down. However, after 72 hours, 88% of the starting C-terminal thioester peptide had reacted. Under these conditions, by-product formation was observed in addition to formation of the desired reaction product. Nevertheless, after 72 hours, HPLC analysis of the reaction mixture estimated that 30-40% of the reaction product was the desired ligation reaction product (N-methoxy peptide amide).

  The reaction of O-methylhydroxylamine with recombinant C-terminal thioester protein was also investigated. As described in Example 2, thiol-mediated cleavage of the fusion protein Grb2-SH2-GyrA intein-CBD produced recombinant Grb2-SH2 as a C-terminal mercaptoethanesulfonic acid thioester derivative. This recombinant C-terminal thioester protein was reacted with 100 mM O-methylhydroxylamine at pH 7.5. Analysis of the reaction mixture after 18 hours by HPLC and ESMS showed that all of the C-terminal thioester protein was completely converted into two protein species. These two types of protein derivatives include a desired ligation reaction product, that is, Grb2-SH2 (expected mass 12067 Da, observed mass 12067 Da) whose C-terminal is N-methoxyamide, and an oxidized form (observed mass) of the desired reaction product. 12084 Da). By-products corresponding to hydrolysis of the C-terminal protein thioester were not observed. Thus, all of the recombinant C-terminal thioester proteins were chemoselectively linked to O-methylhydroxylamine via a reaction that specifically forms an amide bond at the C-terminus of the protein. That is, this reaction resulted in site-specific C-terminal labeling of the recombinant protein.

Example 2 Generation of Grb2 SH2 recombinant protein (recombinant C-terminal hydrazide Grb2 SH2 protein) with C-terminal hydrazide (i) Recombinant C-terminal hydrazide protein by selective cleavage of protein-intein fusion with hydrazine The SH2 domain of the adapter protein Grb2 was selected as a model system to investigate the production capacity and (ii) their subsequent use in ligation / labeling reactions.
Sequence of human Grb2 SH2 domain HPW FFGKIPRAKA EEMLSKQRHD GAFLIRES APGDFSLSVK FGNDVQHFKV LRDGAGKYFL WVVKFNSLNE LVDYHRSTSV SRNQQIFRDQ

Expression of Grb2-SH2 domain-GyrA intein fusion The DNA sequence encoding the SH2 domain of human Grb2 with an extra glycine residue at the C-terminus was cloned into the pTXB1 expression plasmid (NEB). This vector pTXB1 _{Grb2-SH2 (Gly)} has the Grb2 SH2 domain linked to the N-terminus of the GyrA intein via a glycine residue, ie fused to the N-terminus of the chitin binding domain region (CBD). Encodes a fusion protein. E. coli cells were transformed with this plasmid, grown to mid-log phase in LB medium, and protein expression was induced with 0.5 mM IPTG for 4 hours at 37 ° C. After centrifugation, the cells were resuspended in lysis buffer (0.1 mM EDTA, 250 mM NaCl, 5% glycerol, 1 mM PMSF, 25 mM HEPES, pH 7.4) and lysed by sonication. The soluble fraction was loaded onto a chitin column pre-equilibrated in lysis buffer. Next, the column was washed with a washing buffer (1 mM EDTA, 250 mM NaCl, 0.1% Triton-X 100, 25 mM HEPES, pH 7.0), and purified Grb2-SH2-GyrA immobilized on chitin beads. -CBD was obtained (FIG. 7).

Cleavage of the Grb2-SH2-GyrA intein fusion by thiol induction produces Grb2-SH2 with a C-terminal thioester To confirm that the intein domain inside the protein is functional, the fusion protein is exposed to thiol Thus, the degree of cleavage by transthioesterification was evaluated. Chitin beads containing immobilized Grb2-SH2-GyrA-CBD were equilibrated in 200 mM NaCl, 200 mM phosphate buffer, pH 7.4. Next, dithiothreitol (DTT) or 2-mercaptoethanesulfonic acid (MESNA) is added to the beads in 200 mM NaCl, 200 mM phosphate buffer at pH 7.4 to give a final thiol concentration of 100 mM or 120 mM, respectively. A 50% slurry was obtained. The mixture was then shaken at room temperature and an aliquot was analyzed by SDS-PAGE. After 48 hours, the supernatant was separated from the reaction and subsequently analyzed by HPLC and ESMS.

  Whether the Grb2-SH2-GyrA intein-CBD fusion was treated with DTT or MESNA, the fusion protein was cleaved into two protein species (FIG. 7). The molecular size of the two fragments corresponds to the molecular size of the Grb2-SH2 and GyrA-intein fusions, indicating that cleavage occurred at the SH2-intein junction. Cleavage of the fusion protein precursor released the SH2 domain into the supernatant, whereas the GyrA intein-CBD moiety remained immobilized on the chitin beads. After cleaving with both DTT or MESNA, ESMS analysis of the supernatant confirmed that Grb2-SH2 was produced as a derivative with the expected C-terminus of DTT or MESNA being a thioester, respectively.

  The expected mass of Grb2-SH2 having a DTT-C terminal thioester was 12173.9 Da, and the observed mass was 12173.5 Da. The expected mass of Grb2-SH2 having a MESNA-C terminal thioester was 12162.0 Da, and the observed mass was 12163.0 Da.

Cleavage of the Grb2-SH2-GyrA intein fusion by induction of hydrazine generates Grb2-SH2 with a C-terminal hydrazide We have determined that the thioester bond between Grb2-SH2 and GyrA intein in the fusion protein precursor Hypothesized that it is cleaved by hydrazine. If a hydrazine chemoselective reaction occurs at the thioester moiety linking Grb2 SH2 to the intein, the Grb2-SH2 domain should be released into the supernatant as its corresponding C-terminal hydrazide derivative. Therefore, the chitin beads containing immobilized Grb2-SH2-GyrA-CBD were equilibrated in 200 mM NaCl, 200 mM phosphate buffer at pH 7.4, and hydrazine monohydrate was added to the same buffer. A 50% slurry with a final hydrazine concentration of 200 mM was obtained. The mixture was then shaken at room temperature and analyzed by SDS-PAGE (Figure 8). After 20 hours, the supernatant was removed and analyzed by HPLC and ESMS.

  When the Grb2-SH2-GyrA intein-CBD fusion was treated with hydrazine, the fusion protein was cleaved into two species. The molecular size of the two fragments analyzed by SDS-PAGE corresponded to the Grb2-SH2 and GyrA-intein fusions, indicating that cleavage occurred at the unique thioester bond between the SH2 and intein domains . Cleavage of the fusion protein precursor released the SH2 domain into the supernatant, whereas the GyrA intein-CBD moiety remained immobilized on the chitin beads. HPLC and ESMS analysis of the supernatant after cleavage confirmed that a single protein species was generated corresponding to the C-terminal hydrazide derivative of Grb2-SH2. The expected mass of Grb2-SH2C terminal hydrazide was 12051.7 Da and the observed mass was 12053.0 Da (FIG. 9).

  After 20 hours of reaction, Grb2-SH2C-terminal hydrazide was isolated from the supernatant by (i) RPHPLC followed by lyophilization or (ii) gel filtration. In this latter method, the reaction solution of Grb2-SH2C-terminal hydrazide was placed on a Superdex peptide column (Amersham Biosciences) and eluted with a running buffer consisting of 50 mM sodium acetate at pH 4.5. This gave a solution of purified Grb2-SH2C terminal hydrazide dissolved in 50 mM sodium acetate at pH 4.5. The solution was concentrated using a Centricon filter (3000 MWCO), then snap frozen and stored at −20 ° C. until use.

  A sample of purified and lyophilized Grb2-SH2C-terminal hydrazide (100 μg) was treated with protease Lys-C (5 μg) dissolved in 100 mM ammonium bicarbonate buffer (100 μL) at pH 8.2. After overnight incubation at 30 ° C., the reaction was lyophilized and analyzed by MALDI mass spectrometry. The observed mass of the C-terminal proteolytic fragment (FNSLNELVDYHRSTSVSRNQQIFLRRDIEQVPQQPTG) corresponds to the desired C-terminal hydrazide derivative (the expected mass of the proteolytic fragment whose C-terminal is hydrazide is 4229 Da, the observed mass is 4231 Da).

Example 3 Generation of Recombinant Maltose Binding Protein with C-Terminal Hydrazide As a further demonstration of the described method for generating recombinant C-terminal hydrazide proteins by selective cleavage of protein-intein fusions with hydrazine The production of C-terminal hydrazide derivatives of maltose binding protein (MBP) was investigated.
Array MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAAT of human MBP used ENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRGTLEG

Expression of MBP-Sce VMA intein fusion Expression vector pMYB5 (New England Biolabs) is fused at the N-terminus to the Sce VMA intein, ie, the N-terminus of the chitin binding domain (CBD) to facilitate purification. Encodes a fusion protein comprising a maltose binding protein (sequence above) fused to

  E. coli cells were transformed with this plasmid, grown to mid-log phase in LB medium, and protein expression was induced with 0.5 mM IPTG for 4 hours at 37 ° C. After centrifugation, the cells were resuspended in lysis buffer (0.1 mM EDTA, 250 mM NaCl, 5% glycerol, 1 mM PMSF, 25 mM HEPES, pH 7.4) and lysed by sonication. The soluble fraction was loaded onto a chitin column pre-equilibrated in lysis buffer. Next, the column was washed with a washing buffer (1 mM EDTA, 250 mM NaCl, 0.1% Triton-X 100, 25 mM HEPES, pH 7.0), and purified fusion protein (MBP-) immobilized on chitin beads was washed. VMA-CBD) was obtained.

Cleavage of MBP-VMA intein fusion protein by induction of thiol to generate MBP with thioester at the C-terminus To confirm that the intein domain within MBP-VMA-CBD is functional, the fusion protein is 2-mercapto The extent of cleavage by transthioesterification was evaluated by exposure to ethanesulfonic acid (MESNA). Chitin beads containing immobilized MBP-VMA-CBD were equilibrated in 200 mM NaCl, 200 mM phosphate buffer at pH 7.4. Next, MESNA was added to the beads in 200 mM NaCl, 200 mM phosphate buffer at pH 7.4 to obtain a 50% slurry with a final thiol concentration of 120 mM. The mixture was then shaken at room temperature and an aliquot was analyzed by SDS-PAGE. After 48 hours, the supernatant was separated from the reaction and subsequently analyzed by HPLC and ESMS.

  When the MBP-VMA-CBD fusion was treated with MESNA, the fusion protein was cleaved into two protein species. The molecular size of the two fragments corresponds to the molecular size of the MBP and VMA-CBD parts, indicating that cleavage occurred at the MBP-VMA intein junction. Cleavage of the fusion protein precursor liberated MBP into the supernatant, whereas the VMA-CBD moiety remained immobilized on the chitin beads. This was confirmed by ESMS analysis of the cleaved supernatant containing one protein species. The expected mass of MBP whose C-terminal is MESNA thioester was 43064 Da, and the observed mass was 43098 Da.

Generation of MBP with hydrazide at C-terminus by cleavage of MBP-VMA intein fusion protein by induction of hydrazine The chitin beads containing immobilized MBP-VMA-CBD were placed in 200 mM NaCl, 200 mM phosphate buffer at pH 7.4. And hydrazine monohydrate was added in the same buffer to give a 50% slurry with a final hydrazine concentration of 200 mM. The mixture was then shaken at room temperature and analyzed by SDS-PAGE and HPLC and ESMS.

  After 20 hours of reaction, C-terminal hydrazide MBP was isolated from the supernatant by (i) RPHPLC followed by lyophilization or (ii) gel filtration. In this latter method, a reaction solution of C-terminal hydrazide MBP was placed on a Superdex peptide column (Amersham Biosciences) and eluted with a running buffer consisting of 50 mM sodium acetate buffer at pH 4.5. This gave a solution of purified C-terminal hydrazide MBP dissolved in 50 mM sodium acetate buffer at pH 4.5. The protein solution was concentrated using a Centricon filter (3000 MWCO), then snap frozen and stored at −20 ° C. until use.

  When the MBP-VMA-CBD fusion was treated with hydrazine, the fusion protein was cleaved into two species. The molecular sizes of the two fragments analyzed by SDS-PAGE correspond to the MBP and VMA-CBD moieties, indicating that cleavage occurred at the unique thioester bond between the MBP-VMA intein domains. Cleavage of the fusion protein precursor liberated MBP into the supernatant, whereas the VMA-CBD moiety remains immobilized on the chitin beads. HPLC and ESMS analysis of the supernatant after cleavage confirms the generation of a single protein species with an observed mass of 42988 Da. The expected mass difference between the C-terminal MESNA thioester derivative of the protein and the corresponding C-terminal hydrazide is 111 Da. The observed mass of MBP C-terminal MESNA thioester was found to be 43098 Da. Therefore, the product of MBP-VMA-CBD by hydrazine cleavage was 110 Da less, indicating that the desired MBP C-terminal hydrazide derivative was formed.

Example 4 Ligation of Peptides and Labels Containing Aldehydes and Ketones to a Recombinant Protein Containing Hydrazide at the C-Terminal: Ligation of Synthetic Peptide c-myc to Recombinant Grb2 SH2 Domain It was hypothesized that the recombinant protein C-terminal hydrazide produced by hydrazine treatment of the fusion precursor can be modified site-specifically by chemoselective ligation using peptides and labels containing aldehydes and ketones. To demonstrate such a method, the ability of synthetic peptides containing ketones to link with the Grb2-SH2C-terminal hydrazide generated as described above was investigated. It was synthesized synthetic peptides GEQKLISEEDL _--NH2 corresponding to c-myc epitope sequence. At this time, pyruvic acid was coupled to the amino terminus of the peptide as the final step of assembly. The peptide _(CH 3 referred to as COCO-myc) was purified to a purity of more than 95% by RPHPLC, lyophilized (expected monoisotopic mass of ESMS has 1328.6Da; observed mass is 1328.6Da).

A sample of CH ₃ COCO-myc peptide was dissolved in 100 mM sodium acetate buffer at pH 4.5 to give a peptide concentration of 4 mM. Next, this peptide solution (100 μL) was added to a fixed amount (about 250 μg) of the lyophilized Grb2-SH2C-terminal hydrazide protein, and the reaction was observed by SDS-PAGE (FIG. 10). Also, as a control, CH ₃ COCO-myc was incubated with a protein similar in size to cytochrome C, ie Grb2-SH2, but without the hydrazide functional group.

The results of SDS-PAGE analysis show that the CH ₃ COCO-myc peptide is Grb2-SH2C, as shown by the conversion of Grb2-SH2C-terminal hydrazide to a higher molecular weight protein species (approximately 1000-2000 Da larger). It shows that it was actually linked to the terminal hydrazide. After 24 hours, the reaction is substantially complete and the reaction product appears stable. On the other hand, no change in cytochrome C over time was observed, ie no ligation occurred, demonstrating that the ligation reaction occurred at the C-terminal hydrazide functional group of Grb2-SH2.

After 96 hours of reaction, the product of the Grb2-SH2 ligation reaction was isolated by HPLC and characterized by ESMS. Via hydrazone bond formation, If there 'chemoselective ligation of _CH 3 COCO-myc to Grb2-SH2C terminal hydrazide should product expected mass is 13363.7Da is obtained. The observed mass of the product was 13364.1 Da, indicating that the desired ligation product was formed.

Example 5 Ligation of peptide and label containing aldehyde and ketone to a recombinant protein containing hydrazide at the C-terminus (recombinant C-terminal hydrazide protein): Fluorescein labeling of Grb2-SH2 In this example, an intein fusion protein precursor Recombinant C-terminal hydrazide derivative of Grb2-SH2 produced by hydrazine cleavage of the body was reacted with a fluorescein derivative containing ketone to perform site-specific fluorescent labeling of the protein.

In order to facilitate fluorescent labeling of recombinant proteins whose C-terminus is hydrazide according to the method described, the fluorophore must contain an appropriate reactive group for ligation, ie an aldehyde or ketone functional group. is there. For this purpose, a fluorescein derivative containing a pyruvoyl moiety was synthesized. First, Fmoc-Lys (Mtt) -OH was coupled to the rink amide resin and the Mtt group was removed by standard procedures (1% TFA, 4% triisopropylsilane in dichloromethane). Next, 5 (6) -carboxyfluorescein was coupled to the ε-amino group of lysine. Next, the Fmoc group was removed, and pyruvic acid was bound to the free α-amino group of lysine. After cleavage from the resin, the desired fluorescein derivative _(referred to as CH 3 COCO-Lys (Fl) . See Figure 11) was purified to a purity by RPHPLC exceeds 95%, and then lyophilized (the ESMS expected mono Isotopic mass is 576.2 Da; observed monoisotopic mass is 576.0 Da).

To confirm the reactivity of CH ₃ COCO-Lys (Fl) with C-terminal hydrazide peptides and proteins, the reaction of CH ₃ COCO-Lys (Fl) with the small synthetic C-terminal hydrazide peptide SLAYG-NHNH ₂ was investigated. . CH ₃ COCO-Lys with the sample and SLAYG-NHNH ₂ peptide were both dissolved in 100mM sodium acetate buffer pH4.5 for (Fl), to a final concentration of 0.3mM and 2mM, respectively. After 20 hours incubation at room temperature, the reaction was considered complete as determined by RPHPLC analysis. All react with most of the _CH 3 COCO-Lys of the starting (Fl) has occurred a single product. Its mass corresponds to the desired ligation product, ie the combination of two reactants via hydrazone bond formation (ESMS expected monoisotopic mass is 1079 Da; observed mass is 1080 Da).

Since the specific reaction of CH ₃ COCO-Lys (Fl) with hydrazide-containing peptides was confirmed, site-specific labeling of the recombinant C-terminal hydrazide Grb2 SH2 (generated by hydrazine cleavage of Grb2 SH2-GyrA-CBD) This fluorescein derivative was used.

Two complementary methods were used to purify the Grb2 SH2 C-terminal hydrazide from the fusion protein cleavage reaction (Example 2). The purified protein was isolated as a lyophilized solid or in a solution of 50 mM sodium acetate buffer at pH 4.5. The latter buffer system was chosen because the pH is suitable for hydrazone bond formation reactions. A fixed amount (250 μg, 200 μL) of Grb2 SH2 C-terminal hydrazide dissolved in 50 mM sodium acetate at pH 4.5 was added directly to the sample of CH ₃ COCO-Lys (Fl) to bring the final concentration of the fluorophore to about 0. 3 mM. The reaction was incubated at room temperature and observed by SDS-PAGE. As a control, CH ₃ COCO-Lys (Fl) was incubated under the same conditions with a protein similar in size to cytochrome C, ie Grb2-SH2, but without the hydrazide functional group.

The results of the SDS-PAGE analysis indicate that the Grb2-SH2C-terminal hydrazide has been converted to a single protein species (approximately 1000-2000 Da larger) with apparently increased molecular weight, as shown in CH ₃ COCO-Lys. (Fl) is actually linked to the Grb2-SH2C terminal hydrazide (FIG. 12). After SDS-PAGE analysis of the reaction, gel fluorescence imaging confirmed that the newly formed reaction product contained a fluorescein label and that the reaction was clean and a single fluorescent protein product. Only the formation was confirmed (FIG. 14). After 24 hours, the reaction is substantially complete and the reaction product appears stable under these conditions.

  On the other hand, no change in cytochrome C was observed over the course of the experiment, ie no ligation occurred (FIG. 13) and no formation of any fluorescent protein product (FIG. 14). This therefore demonstrates that the ligation reaction occurs at the C-terminal hydrazide functional group of Grb2 SH2, resulting in a site-specific C-terminal fluorescent label of the recombinant protein. After 48 hours of reaction, the product obtained from the ligation reaction with Grb2 SH2 was isolated by HPLC. From the mass of the product measured by ESMS, it was confirmed that one fluorescein group was added to the protein.

In another embodiment, dissolved in 100mM sodium acetate the Grb2 SH2C terminal hydrazide was directly lyophilized pH _4.5, was added to CH 3 COCO-Lys (Fl) . Although some protein precipitation was observed, the soluble fraction of the protein reacted with _CH 3 COCO-Lys (Fl) in the expected manner described above.

In an alternative strategy, a lyophilized sample (250 μg) of Grb2 SH2 C-terminal hydrazide was dissolved in 0.1% TFA (200 μL) containing 40% aqueous acetonitrile. Next, this solution was added to a sample of _{CH 3 COCO-Lys (Fl)} , the final concentration of the fluorophore was about 0.3 mM. The solution was incubated at room temperature and the reaction was analyzed periodically. SDS-PAGE analysis showed that the labeling reaction occurred cleanly and rapidly under these conditions (FIG. 15). The Grb2 SH2C-terminal hydrazide has been converted to a single protein species with a clearly increased molecular weight expected to be the molecular weight of the desired product, and this newly formed protein is visualized under a UV lamp. A green fluorescent color was exhibited. From the ESMS of this reaction product, it was confirmed that one fluorescein molecule was added to the protein. The reaction is substantially complete after 4 hours, and prolonged incubation appears to be detrimental to ligation product formation.

Example 6 Ligation of peptides and labels containing aldehydes and ketones to recombinant proteins containing hydrazide at the C-terminus: Fluorescein labeling of MBP As a further illustration, MBP is site-specifically directed by fluorescein using the method described. C-terminally labeled. Lyophilized recombinant C-terminal hydrazide MBP sample (250 μg) (generated by hydrazine cleavage of MBP-VMA-CBD fusion protein precursor) was dissolved in 40% aqueous acetonitrile (200 μL) containing 0.1% TFA. It was. Next, this solution was added to a sample of _{CH 3 COCO-Lys (Fl)} , the final concentration of the fluorophore was about 0.3 mM. The reaction was then incubated at room temperature and analyzed periodically by SDS-PAGE.

  SDS-PAGE analysis showed that a fluorescein labeling reaction occurred under these conditions, as indicated by the formation of a single green fluorescent species having a molecular weight of about 42 KDa. The results of MALDI analysis of the reaction mixture after 48 hours were consistent with the addition of one fluorescein molecule to MBP.

  In summary, the present invention provides a novel method for protein ligation and protein labeling. These allow protein fragments derived by both synthesis and recombination to be efficiently linked together in a regioselective manner. This therefore allows large proteins to be constructed from a combination of synthetic and recombinant fragments, and allows site-specific modification of any size protein in an unprecedented manner. Considering that 30,000 human genes yield hundreds of thousands of different protein species through post-translational modifications, this is very important for bioscience and biomedicine and drug discovery. Such proteins that have been post-translationally modified cannot be evaluated by current recombinant techniques.

  The application of such protein ligation technology can be used for protein-based approaches and protein therapies, and in de novo design (new design), much of the bioscience and biomedicine that has not been possible before. Can open up new ways.

  All documents referred to herein are hereby incorporated by reference. Various modifications and variations of the described embodiments of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in terms of particular preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to these specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be encompassed by the present invention.

FIG. 2 schematically shows the general principle of chemical ligation. It is a figure which shows roughly the mechanism of protein splicing. It is a figure which shows the production | generation of the recombinant protein whose C terminal is a thioester. FIG. 2 shows ligation of protein and peptide thioesters with hydrazine and aminooxy-containing units such as labels, peptides, and proteins. FIG. 4 shows synthesis and production of recombinant peptide hydrazide for ligation with thioester-containing molecules. Note that the peptide or label is an acyl substituent of the thioester. FIG. 5 shows the production of recombinant peptide hydrazide for ligation with molecules containing aldehydes and ketones. It is a photograph which shows the result of SDS-PAGE analysis of Grb2-SH2-GyrA-CBD (immobilized on chitin beads) treated with DTT and MESNA. Molecular weight marker (lane 1); purified Grb2-SH2-GyrA-CBD immobilized on chitin beads (lane 4). Grb2-SH2-GyrA-CBD treated with 100 mM DTT (lanes 5 and 7) or 120 mM MESNA (lanes 8 and 10). Both the entire reaction slurry (lanes 5 and 8) and the reaction supernatant (lanes 7 and 10) were analyzed. It is a photograph which shows the result of the SDS-PAGE analysis of Grb2-SH2-GyrA-CBD (immobilized on chitin beads) treated with hydrazine. Molecular weight marker (lane 1); purified Grb2-SH2-GyrA-CBD (lane 2) immobilized on chitin beads after treatment with phosphate buffer alone for 20 hours. Grb2-SH2-GyrA-CBD treated with 200 mM hydrazine dissolved in phosphate buffer for 20 hours. The entire reaction slurry was analyzed. It is a graph which shows the ESMS spectrum of the Grb2-SH2 derivative whose C terminal is hydrazide. A ketone-containing _CH 3 COCO-myc peptide synthesis is a photograph showing the results of SDS-PAGE analysis of the reaction with the C-terminal hydrazide Grb2-SH2 and cytochrome C. Molecular weight marker (lane 1); Grb2-SH2 (lane 2) in which the C-terminus is a DTT thioester. At time t = 0 (hour) (lane 3), t = 24 (hour) (lane 4), t = 48 (hour) (lane 5), t = 72 (hour) (lane 6), the C-terminal is hydrazide Reaction of Grb2-SH2 with CH ₃ COCO-myc. Cytochrome C and CH at time t = 0 (hour) (lane 7), t = 24 (hour) (lane 8), t = 48 (hour) (lane 9), t = 72 (hour) (lane 10) ₃ Reaction with COCO-myc. CH ₃ illustrates the structure of COCO-Lys (Fl). The regioisomer of 5-carboxyfluorescein is shown. The reaction between _CH 3 COCO-Lys (Fl) and C-terminal hydrazide Grb2-SH2 in a 50mM sodium acetate buffer at pH4.5 is a photograph showing the results of SDS-PAGE analysis. Molecular weight marker (lane 1); Grb2-SH2 (lane 2) in which the C-terminus is hydrazide. Grb2-SH2 and CH ₃ COCO-Lys in which C-terminal is hydrazide at time t = 4 (hour) (lane 3), t = 24 (hour) (lane 4), and t = 48 (hour) (lane 5) Reaction with (Fl). _CH 3 COCO-Lys in a 100mM sodium acetate buffer pH4.5 and (Fl) the reaction of the cytochrome C is a photograph showing the results of SDS-PAGE analysis. Molecular weight marker (lane 1); cytochrome C (lane 2). Reaction of cytochrome C with CH ₃ COCO-Lys (Fl) at time t = 4 (hour) (lane 3), t = 24 (hour) (lane 4), t = 48 (hour) (lane 5). FIG. 6 shows the results of SDS-PAGE analysis of the reaction of CH ₃ COCO-Lys (Fl) with Grb2-SH2 having C-terminal hydrazide and cytochrome C in 50 mM sodium acetate buffer at pH 4.5. It is a photograph. (A) Staining of total protein in the gel. Prior to this Coomassie staining (A), the gel was imaged against green fluorescence (B). Molecular weight marker (lane 1); Grb2 SH2 having C-terminal hydrazide (lane 2); time t = 4 (hour) (lane 3), t = 24 (hour) (lane 4), t = 48 (hour) ( reaction in lane 5), and C-terminal hydrazide Grb2 SH2 and _{CH 3 COCO-Lys (Fl)} . Cytochrome C (lane 6); cytochrome C and CH ₃ COCO-Lys at time t = 4 (hour) (lane 7), t = 24 (hour) (lane 8), t = 48 (hour) (lane 9) Reaction with (Fl). Is a photograph showing a _CH 3 COCO-Lys (Fl) and a result of the reaction of the C-terminal hydrazide Grb2 SH2 was SDS-PAGE analysis in 0.1% TFA-containing 40% aqueous acetonitrile. 4 hours after the reaction (lane 1), 24 hours (lane 2), 48 hours (lane 3), and Grb2-SH2 in which the C-terminus is hydrazide (lane 4)

Claims (26)

a) providing a first oligopeptide having a reactive moiety , wherein the reactive moiety is a hydrazine moiety, a hydrazide moiety, or an aminooxy moiety ;
b) providing a second oligopeptide having an activated ester moiety , wherein the activated ester moiety is a thioester moiety, a phenol ester moiety, a hydroxysuccinimide moiety, or an O-acylisourea moiety ;
c) reacting the reactive moiety of the first oligopeptide with the activated ester moiety of the second oligopeptide, via the linking moiety of formula I, formula II, or formula III Forming an oligopeptide product in which the peptide and the second oligopeptide are linked together.

2. The method of claim 1, wherein the activated ester moiety is a terminal activated ester moiety, the terminal activated ester moiety is a thioester, and the second oligopeptide is a thioester acyl substituent. 3. The method of claim 2, wherein the second oligopeptide is generated by thiol reagent dependent cleavage of a precursor molecule comprising a second oligopeptide fused to the intein domain at the N-terminus. a) providing a first oligopeptide having a reactive moiety , wherein the reactive moiety is a hydrazine moiety, a hydrazide moiety, or an aminooxy moiety ;
b) i) providing an oligopeptide precursor molecule comprising a second oligopeptide fused to the intein domain at the N-terminus;
(Ii) subjecting the precursor molecule to thiol reagent dependent cleavage to produce a second oligopeptide molecule having a thioester moiety at the C-terminus;
c) reacting the reactive part of the first oligopeptide with the second oligopeptide molecule, via the linking moiety of formula I, II, or III, the first oligopeptide and the second oligo Forming an oligopeptide product in which the peptide is linked to form an oligopeptide product.

5. A method according to any one of claims 1 to 4, wherein the reactive moiety is an aminooxy moiety and the activated ester moiety is a thioester. Wherein the first oligopeptide, with hydrazine, is prepared by reaction of the precursor molecule comprising an oligopeptide precursor that is fused to the intein domain at the N-terminus via a thioester moiety, any of claims 1 to 4 the method according to one paragraph or. a) providing a first oligopeptide having a reactive moiety that is a hydrazine moiety, a hydrazide moiety, or an aminooxy moiety;
b) providing an oligopeptide precursor molecule comprising a second oligopeptide fused to the intein domain at the N-terminus;
(C) reacting the reactive portion of the first oligopeptide with the oligopeptide precursor molecule to form the first oligopeptide and the second through a linking moiety of formula I, formula II, or formula III. Forming an oligopeptide product in which the oligopeptide is linked to a method of producing an oligopeptide product.

Wherein the first oligopeptide or the second oligopeptide is a recombinant oligopeptide, the first oligopeptide and the other of said second oligopeptide is a synthetic polypeptide, the 請 Motomeko 1-7 The method according to any one of the above. The method according to any one of claims 1 to 7 , wherein the first oligopeptide and the second oligopeptide are recombinant oligopeptides. The method according to any one of claims 1 to 7 , wherein the first oligopeptide and the second oligopeptide are synthetic oligopeptides. Providing a protein component child comprising (a) an oligopeptide which is fused to the intein domain at the N-terminus,
(B) reacting the protein molecule with hydrazine such that the intein domain is cleaved from the oligopeptide to produce a protein hydrazide. The steps of the method (c) is carried out at a pH in the range of pH 6.5-7.5, the method according to any one of claims 1 to 10. a) providing a first oligopeptide having an aldehyde or ketone moiety;
b) providing an oligopeptide precursor molecule comprising a second oligopeptide fused to the intein domain at the N-terminus;
c) reacting said oligopeptide precursor molecule with hydrazine to produce an oligopeptide molecule comprising an oligopeptide intermediate having a hydrazide moiety at the end;
d) The first oligopeptide and the second oligopeptide are linked via a hydrazone linking moiety by reacting the aldehyde or ketone moiety of the first oligopeptide with the hydrazide moiety of the oligopeptide intermediate molecule. Forming an oligopeptide product. The method of making an oligopeptide product. A oligopeptide product produced by the method according to any one of 請 Motomeko 1-13, the said first oligopeptide via a linking moiety having Formula II or Formula III, An oligopeptide product in which two oligopeptides are linked.

a) providing a labeled molecule having a reactive moiety , wherein the reactive moiety is a hydrazine moiety, a hydrazide moiety, or an aminooxy moiety ;
b) providing an oligopeptide having an activated ester moiety , wherein the activated ester moiety is a thioester moiety, a phenol ester moiety, a hydroxysuccinimide moiety, or an O-acylisourea moiety ;
c) reacting the reactive moiety of the labeled molecule with the activated ester moiety of the oligopeptide to link the labeled molecule and the oligopeptide via a linking moiety represented by Formula I, Formula II, or Formula III. Forming a labeled oligopeptide comprising: labeling the oligopeptide.

In step (c), the labeled molecule and the oligopeptide are linked via a linking moiety represented by formula II, wherein the activated ester moiety in step (b) is not a thioester and the activated ester is terminal. The method of claim 15 , wherein the method is an activated ester moiety. a) providing a labeled molecule having an activated ester moiety that is an acyl substituent , wherein the activated ester moiety is a thioester moiety, a phenol ester moiety, a hydroxysuccinimide moiety, or an O-acylisourea moiety. Steps ,
b) providing an oligopeptide having a reactive moiety , wherein the reactive moiety is a hydrazine moiety, a hydrazide moiety, or an aminooxy moiety ;
c) reacting the activated ester moiety of the labeled molecule with the reactive moiety of the oligopeptide to link the labeled molecule to the oligopeptide via a linking moiety represented by Formula I, Formula II, or Formula III. Forming a labeled oligopeptide comprising the steps of:
In step (c), the labeled molecule and oligopeptide are linked via a linking moiety represented by formula II, wherein the activated ester moiety in step (b) is not a thioester and the activated ester is terminally active. A method wherein the esterified ester moiety.

18. The method of claim 17 , wherein the oligopeptide is made by reaction of hydrazine with a precursor molecule comprising an oligopeptide precursor fused to the intein domain at the N-terminus via a thioester moiety. a) providing a label having a reactive moiety , wherein the reactive moiety is a hydrazine moiety, a hydrazide moiety, or an aminooxy moiety ;
b) (i) providing an oligopeptide precursor molecule comprising an oligopeptide fused to the intein domain at the N-terminus;
(Ii) causing a thiol reagent-dependent cleavage of the precursor molecule to produce an oligopeptide molecule having a thioester moiety at the C-terminus;
c) reacting the reactive part of the label with an oligopeptide molecule to form a labeled oligopeptide in which the label and oligopeptide are linked via a linking moiety of formula I, II, or III And a step of labeling the oligopeptide.

18. A method according to any one of claims 15 to 17 wherein the reactive moiety is an aminooxy moiety and the activated ester moiety is a thioester. 20. The method of claim 19 , wherein the reactive moiety is an aminooxy moiety. a) providing a labeled molecule having a reactive moiety , wherein the reactive moiety is a hydrazine moiety, a hydrazide moiety, or an aminooxy moiety ;
b) providing an oligopeptide precursor molecule comprising an oligopeptide fused to the intein domain at the N-terminus;
c) A label in which a reactive part of a label molecule is reacted with an oligopeptide precursor molecule, and the label molecule and the oligopeptide are linked via a linking moiety represented by Formula I, Formula II, or Formula III Forming an oligopeptide product comprising: labeling the oligopeptide.

23. A method according to any one of claims 15 to 22 , wherein step (c) of the method is carried out at a pH in the range of pH 6.5 to 7.5. a) providing a labeled molecule having an aldehyde or ketone moiety;
b) providing an oligopeptide precursor molecule comprising a first oligopeptide fused to the intein domain at the N-terminus;
c) reacting said oligopeptide precursor molecule with hydrazine to produce an oligopeptide molecule comprising an oligopeptide intermediate having a hydrazide moiety at the end;
d) reacting the aldehyde or ketone moiety of the labeled molecule with the hydrazide moiety of the oligopeptide intermediate molecule to produce a labeled oligopeptide product in which the labeled molecule and oligopeptide are linked via a hydrazone linking moiety. Forming the oligopeptide, comprising the step of forming. 25. A method according to claim 13 or claim 24 , wherein the aldehyde or ketone moiety is an [alpha] -diketone or [alpha] -keto-aldehyde group. 26. A labeled oligopeptide produced by the method according to any one of claims 15 to 25 , wherein the labeled oligopeptide is linked to the first oligopeptide via a linking moiety represented by formula II or formula III. A labeled oligopeptide linked to a second oligopeptide.

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